Completing the genetic spectrum influencing coronary artery disease: from germline to somatic variation

Abstract

Genetic and environmental factors influence the development of coronary artery disease (CAD). Genetic analyses of families and the population continue to yield important fundamental insights for CAD. For the past four decades, CAD human genetic research focused largely on the study of germline genetic variation in CAD and its risk factors. The first genes associated with CAD were discovered using basic Mendelian principles and pedigree analysis. Mapping of the human genome and advancement in sequencing technology sparked further discovery of novel genetic associations through exome sequencing and genome wide association analysis in increasingly larger populations. While prior work implicated in situ DNA damage as a feature of atherosclerosis, more recently, somatic mutagenesis in and clonal expansion of haematopoietic stem cells was found to influence risk of CAD. Mutations observed for this condition, termed clonal haematopoiesis of indeterminate potential, frequently occur within epigenetic regulator genes (e.g. DNMT3A, TET2, ASXL1, etc.), which are also implicated in leukaemogenesis. Hypercholesterolaemic mice with Tet2 bone marrow deficiency are predisposed to the development of atherosclerosis that may be partly related to inflammatory cytokines. As the genetic basis of CAD expands from the germline to somatic genome, our fundamental understanding of CAD continues to evolve; these new discoveries represent new opportunities for risk prediction and prevention, and a new facet of cardio-oncology.

Keywords: Clonal haematopoiesis of indeterminate potential; Coronary artery disease; Genetics; Somatic mutations.

Figure 1

Germline and somatic mutagenesis. In germline mutagenesis, a mutation is transmitted from parent to offspring and all subsequent lineages carry it. In somatic mutagenesis, a mutation occurs in a stem cell or replicating differentiated cell leading to mosaicism, and potentially clonal advantage in replication and survival.

Figure 2

Advances in genetic analysis methodology. (A) Linkage analysis is a statistical method that tracks and correlates hereditary phenotype transmission with genetic loci relying on the consequences of genetic recombination between two autosomal chromosomes during meiosis. Finer resolution mapping is possible with larger pedigrees. This schematic depicts an autosomal dominant trait originating from the terminal chromosomal locus in the pedigree father (marked x) and being transmitted to a granddaughter via an affected son and to a grandson and granddaughter via an affected daughter. (B) Sanger sequencing is used to determine the DNA sequence for a locus. Repetitive polymerase chain replication is performed in which a constitutively replicated sequence fragment is randomly terminated with a labelled chain-terminating dideoxynucleotide. Several sequencing fragments of varying lengths and end nucleotides emerge and are separated based on length. The attached base for each length is imaged to yield a sequence of bases. (C) Next generation sequencing refers to a group of methods of large-scale, parallel sequencing using Sanger sequencing principles. This involves fragmenting genomic DNA, attaching adaptors to the newly formed DNA fragments, attaching these adaptors to a solid surface for sequencing, amplifying the fragmented DNA strands in parallel, enriching desired segments, sequencing in parallel using different methods, aligning the sequence reads that result from the pooled results to known genomic reference sequence, and examining the resulting DNA variants. (D) Genome wide association (GWA) involves using large genotyping arrays containing SNPs, each representative of a locus of bases that are generally inherited together due to linkage disequilibrium. DNA from cohorts of individuals affected and unaffected by a trait are genotyped with these arrays and the comparative differences in prevalence of certain mutations are used to statistically determine associations of polymorphisms at a certain locus with the trait of interest.

Figure 3

Prevalence of the most common mutations implicated in clonal haematopoiesis of indeterminate potential (CHIP) in three seminal studies.

Figure 4

Interplay of well-known germline and somatic mutations in atherogenesis. In the liver, cholesterol biosynthesis begins with acetyl-CoA, and intracellular cholesterol levels are regulated with assistance the SREBP2 pathway. Statins interfere with cholesterol synthesis by inhibiting HMG-CoA reductase, leading to a drop in intracellular cholesterol levels, synthesis of more LDL receptors, and increased uptake of LDL cholesterol from the circulation. PCSK9 molecules help regulate the number of LDL receptors on the cell surface by aiding in their uptake from the cell membrane and transport to the lysosome for degradation. Evolocumab and alirocumab inhibit the activity of PCSK9, allowing more LDLR-mediated uptake of serum cholesterol. Sortilin expression, mediated by enhancers at the chromosome 1p13 locus, promotes release of mature VLDL particles into the circulation, PCSK9 secretion, and macrophage lipid accumulation leading to foam cell formation. In the circulation, various forms of lipoproteins combined with lipid molecules transport their contents throughout the body. APOA5 and APOC3 on VLDL particles participate in triglyceride metabolism and inhibit lipoprotein lipase (LPL), respectively. ANGPTL3 also inhibits LPL, leading to increased circulating levels of cholesterol and triglycerides, which are deposited in the endothelium of the vasculature. In the bone marrow, somatic mutations in TET2 lead to hyperproliferative advantage for a subset of haematopoietic pluripotent stem cells (HPSCs), leading to clonal haematopoiesis of indeterminate potential (CHIP). The clonal monocytes that result from further replication produce an abundance of IL-1 beta, which promotes further inflammatory cascades and is in part inhibited by canakinumab. These macrophages adhere to the lipid-rich endothelium and traverse it. Within the vessel wall, mitochondrial dysfunction and generation of ROS leads to oxidation of LDL, generation of more inflammatory cytokines, and further damage to surrounding cells. Macrophages consume this oxidized LDL to become foam cells. Vascular smooth muscle cells (VSMCs) proliferate, damaged cells apoptose, and the atheroma continues to grow.